Primary energy transduction in biological systems is derived from two coupled sources. Oxidative phosphorylation (and substrate phosphorylation) ultimately generate both an ion motive force and cytoplasmic ATP, in reactions where the reactants and products are physically and spatially coupled. Both of these energy sources are used to drive active transport of nutrients across the cytoplasmic membrane. The outer membranes of Gram-negative bacteria present a special problem: They are unenergized and they are impermeant to molecules of greater than 600 Da. In order to obtain vital nutrients, these organisms have developed a sophisticated system whereby energy generated by conventional means (cytoplasmic membrane protonmotive force) can be transduced to transport proteins in the outer membrane for active transport of nutrients into the periplasmic space. This system appears to constitute a new biological paradigm. Using E. coil as a model system, it has become clear that several proteins are involved in this process, at both the cytoplasmic and outer membranes. The central player in this process, TonB, is hypothesized to carry conformationally constrained potential energy, and shuttle back and forth between the two membranes to obtain and then deliver it. In the cytoplasmic membrane ExbB and ExbD are hypothesized to convert Ton B to its energized form and to provide the energy that runs the shuttle. In the outer membrane are proteins with which TonB may dock as well as the outer membrane transporters to which the energy is delivered. In this proposal a further understanding of the molecular details of the energy transduction mechanism are sought. Specifically, we will 1) define the functions of the TonB amino and carboxy termini 2) investigate the energy source for the shuttle 3) continue to define the roles of ExbB and ExbD, and 4) initiate studies, based on the structure of colicin B, to characterize the stages of TonB-dependent colicin infection, starting at the outer membrane.